Reverse-transcription polymerase chain reaction is a method of generating and exponentially amplifying DNA copies of an RNA template. The method has both qualitative and quantitative applications in the field of gene expression. The method allows to both detect and measure the levels of mRNA expressed by an organism.
The principal difficulty with RT-PCR is contamination of RNA preparations with genomic DNA. As admitted by the leading distributor of RNA isolation reagents and technologies, most RNA isolation techniques yield RNA with significant amount of genomic DNA contamination. (Ambion, Austin, Tex., (Life Technologies, Inc.), Technical Bulletin #176 “Avoiding DNA contamination in RT-PCR.”) DNA contamination is especially problematic for RT-PCR, where the smallest amount of contaminating DNA will be exponentially amplified. One method of reducing the amplification of DNA by RT-PCR involves pre-treating the samples with deoxyribonuclease, such as DNase I, see Huang, et al. (1996) Biotechniques 20:1012-1020. Unfortunately, this approach is not without problems. After pre-treatment, the DNase must be completely inactivated in order to prevent digestion of the nascent DNA amplicons in the course of RT-PCR. However, high temperatures necessary for compete inactivation of the DNAse cause degradation of the RNA template. As an alternative to heating, one may chemically remove the DNase by phenol extraction or using various elaborate and costly reagents that remove DNase from the reaction mixture. In summary, the use of DNase is impractical in RT-PCR as it requires multiple additional steps and often threatens the fragile RNA target.
Since the problem of DNA contamination is considered intractable, efforts have been devoted to preventing amplification of the DNA contaminant by RT-PCR. One such strategy takes advantage of the presence of introns in eukaryotic genomic DNA. In mature mRNA, the introns are absent. If the primers are designed to flank an intron, the intron will be absent from the amplicon generated from mRNA. However, the intron will be included in the RT-PCR amplicon generated from the corresponding genomic DNA template. If the intron is sufficiently large, the shorter mRNA sequence (and subsequently the cDNA sequence) will be preferentially amplified, while the genomic DNA will be amplified less efficiently or not at all (Ambion, Tech. Bull. #176). In the worst case scenario, the genomic DNA will be co-amplified with the desired mRNA target, but the two amplicons will be distinguishable by electrophoresis.
Unfortunately, primer design is not always capable of overcoming the problem of DNA contamination. Many PCR tests now involve real-time PCR, a technique that does not include electrophoresis but is able to detect nucleic acids simultaneously with amplification, see U.S. Pat. Nos. 5,994,056 and 5,876,930 and related patents. Without electrophoresis, real-time PCR is not capable of parsing out different-size amplicons generated with the same set of primers. Any real-time PCR probe that detects an mRNA target will inevitably also detect the corresponding genomic DNA contaminant. An mRNA and its corresponding genomic DNA will not be distinguished. Therefore, where introns in the region of interest are too small to preclude amplification of genomic DNA, real-time PCR may not be used.
It is therefore desirable to create a novel method of primer design that will ensure that genomic DNA contaminants are not co-amplified with mRNA during RT-PCR. Such a primer design method would enable quantitative amplification of mRNA targets regardless of the size of the intron present in the amplicon.